Francisella tularensis is a facultative intracellular bacterial pathogen that causes serious and potentially life threatening illness. Because the bacterium grows readily in broth culture, has extraordinarily high infectivity, causes serious morbidity and mortality, and is easily dispersed, it is also considered a potential agent of bioterrorism. While currently available antibiotics are effective in treating tularemia, F. tularensis can be engineered to carry antibiotic resistance genes. For these reasons, new approaches to treatment and prevention of tularemia are needed. However, devising such strategies requires an improved understanding of the cell biology of F. tularensis. At present, very little is known about the interactions between F. tularensis and its host cells or of the pathogenic mechanisms that allow F. tularensis to enter, survive, and multiply within host cells. The main objective of this application is to improve our understanding of the cell biology of F. tularensis. Specifically, our aims are: (a) to examine the adherence, uptake and growth of F. tularensis in human mononuclear phagocytes to confirm it as a model system for further study; (b) to examine the ultrastructural features of the F. tularensis compartment in human macrophages at sequential times after infection; and (c) to define the membrane trafficking interactions of the host cell and the F. tularensis compartment by immunofluorescence and immunoelectron microscopy and by examining the effect of overexpression of wild-type and mutant proteins involved in membrane trafficking. We shall combine techniques of cell biology, molecular biology, and immunoelectron microscopy to accomplish these research objectives. An improved understanding the cell biology of F. tularensis is the first step in understanding its pathogenic mechanisms. Understanding how F. tularensis subverts the host cell membrane trafficking pathways and attains an intracellular compartment that is hospitable for its survival and growth will help guide new strategies for the prevention and treatment of tularemia.